Project Summary M1 aminopeptidases (M1APs) are a diverse family of metalloenzymes distributed in a wide range of organisms ranging from prokaryotes to eukaryotes. They play critical roles in many physiological processes, and have been implicated in various chronic and infectious diseases of humans. Structure and mechanism of the conserved catalytic domain, termed peptidase_M1 domain, have been well studied. As a result, many research groups have begun to assess their potential as therapeutic targets for various diseases. However, targeting M1APs to treat human diseases is complicated because there are nine characterized and closely related M1APs in humans. Thus any chemical probes used in target validation must be carefully designed to ensure a selective targeting against a single M1AP over other family members. Nonetheless, current inhibitor-developing strategies rely heavily on Bestatin derivatives to occupy the highly conserved peptidase_M1 site. Although potent, these inhibitors also inhibit many other essential M1AP members, lead to undesired side effects. Recently, a novel ERAP1 C-terminal domain structure, now classified as the ERAP1_C like domain, has been shown by our lab to bind to the substrate?s C-terminus. This ERAP1_C domain appears in all M1AP structures, including endoplasmic reticulum aminopeptidase 1 (ERAP1) and insulin-regulated aminopeptidase (IRAP). Curiously, although with a similar fold, the ERAP1_C domain exhibits a substantial sequence variation among M1AP members, bringing about the exciting possibility to uncover specificity sites for selective inhibitor targeting. To test this hypothesis, we propose in this R15 project to study specific interactions between M1APs and their corresponding substrates, with a focus on the recognition and allosteric mechanism of this novel ERAP1_C domain. Three specific aims are proposed in this application: 1) investigate the molecular ruler mechanism and substrate specificity of ERAP1; 2) explore the structure-function relationship of IRAP; and 3) probe the determinants of substrate specificities among different aminopeptidases. To achieve these goals, we designed experiments that will use specific catalytic assays, mechanistic studies, mutational analyses, x-ray crystallography, and bioinformatics analyses. The proposed research will provide insights into the details of substrate selectivity and the allosteric mechanism to activate peptide cleavage, and is thus critical to improve our capability to develop selective inhibitors to fight against pathogens or chronic diseases. Furthermore, this AREA research will provide continuous research opportunities for undergraduate and graduate students at UMass Lowell, and help the University to enhance its research environment.